Dissertations / Theses on the topic 'Pharmaceutical microbiology'

To meet the demands of political, ethical and scientific pressures on animal testing, research into possible alternatives is required. Data obtained with animal models often cannot be related to humans. Testing with current cell-based assays, microdosing and pharmacokinetic models contribute to reducing animal testing and improving the drug development process. Micro-fabrication and rapid prototyping techniques offer potential solutions to reduce the need for animal toxicity testing. The aim of this research was to develop biological platforms for in vitro toxicity testing to provide physiologically relevant, high-throughput solutions to reduce animal testing. This was achieved by investigating and integrating microfabrication methods of microfluidics, dielectrophoresis and additive manufacturing. Three approaches were taken: (i) micro-pattern protein arrays for primary hepatocyte cell culture enclosed within microfluidics devices for high-throughput toxicity testing. It was observed that hepatocytes attached to the micro-pattern within microfluidics and maintained viability, however liver specific functions observed by florescence assays, the P450 enzymes, were observed to be reduced compared to Petri dish conditions. (ii) A biomimetic dielectrophoretic cell patterning technique to form liver lobule-like tissue structures within agar on a paper substrate was developed for toxicity testing. Observation of these biomimetic micro liver structures showed high viability (80-90%) and an increase in liver specific function marker albumin protein (20%) compared to control samples after 48 hours. (iii) Rapid prototyping methods were explored with regard to fabrication of microfluidic chips for the automated trapping, imaging and analysis of zebrafish embryos. Monolithic microfluidic chips for zebrafish were developed to be suitable for optical based toxicity assays. The biocompatibility of 3D printed materials was investigated. A method to render the photopolymer Dreve Fototec 7150 compatible with zebrafish culture was observed to provide 100% viability. Future development of this research will aim to (i) develop the liver lobule-like system to use layers of multiple cell types to form complex micro-liver models using additive manufactured microfluidic systems for toxicity testing. (ii) Automation of zebrafish handing using additive manufactured microfluidic devices for in-situ analysis of dechorionated zebrafish for high-throughput toxicity studies.

El control microbiològic és essencial en la indústria farmacèutica ja que es tracta d’un indicador de la seguretat dels productes. Els mètodes microbiològics utilitzats a les companyies farmacèutiques per analitzar l’ambient del procés de fabricació així com els productes finals estan basats en mètodes de cultiu tradicional. La tecnologia d’aquests mètodes manca de precisió comparada amb les tecnologies més modernes de detecció i identificació microbiològica. Cada vegada, els requeriments dels controls microbiològics de productes i processos són més estrictes, el que fa que es necessiten mètodes cada vegada més ràpids i precisos. Dintre d’aquest context s’han desenvolupat els mètodes de microbiologia ràpida (RMM) per a la seva implantació a la industria farmacèutica, avalada per les diferents agències reguladores. Les diferents tecnologies en les quals es basen els RMM es coneixen a l’àmbit acadèmic des de fa dècades, no obstant la seva implantació i validació a les indústries farmacèutiques és relativament recent. La implementació de noves metodologies a l’àmbit farmacèutic està regulada per la normativa de bones practiques de fabricació (BPF), el qual va associat a una adequada validació de les tècniques. Aquesta tesi descriu la implementació de RMM al control microbiològic del procés de fabricació i productes a l’empresa Laboratoris Reig Jofré (RJF). El principal objectiu ha estat dividit en 3 blocs individuals: implementació d’un programa d’identificació microbiana per als aïllats del procés de fabricació i productes; implementació d’un mètode de detecció de microorganismes a l’aire de sales classificades per fabricació asèptica basat en fluorescència induïda per làser i l’ avaluació d’un sistema basat en citometria en fase sòlida per la detecció de microorganismes en productes filtrables. En general, els RMM implementats han contribuit a obtenir resultats de manera més ràpida, el qual permet mitigar el risc de contaminació en el mateix moment que ha sigut detectat. Aquest projecte assenta les bases per futures aplicacions de RMM en el procés de fabricació farmacèutica.
Microbiological testing plays an essential role in the Pharmaceutical Industry as it is an indicator of safety in drug products. The microbiological methods used in the pharmaceutical companies for testing the environment of the manufacturing process as well as the final products are based on traditional culture methods. These methods, although being appropriate for their intended use, rely on century-old technology that lacks accuracy when compared to most up-to-date methodologies for microbial detection and identification. Every time the requirement of microbiological testing of products and processes increases, arising the need of faster and more accurate methods. In this paradigm, rapid microbiological methods (RMM) are developed for their implementation in the pharmaceutical industry encouraged by regulatory agencies. The different technologies in which rapid microbiological methods are based have been known in the academic field for decades, however their implementation and validation in the pharmaceutical industries is relatively recent. Implementation of new methodologies in a pharmaceutical environment ruled under Good Manufacturing Practice (GMP) guidelines needs proper validation of the techniques involved. This thesis describes the implementation of RMM in the microbial monitoring of the pharmaceutical manufacturing process and products in Reig Jofré Laboratories (RJF). The main objective has been divided into three different blocks: implementation of a microbial identification program for the isolates found in the production process and products; implementation of a laser-induced fluorescence system for the detection of airborne microorganisms in cleanrooms for aseptic processing and evaluation of a solid-phase cytometry system for the detection of microorganisms in filterable products. In general, the RMM implemented have contributed to obtain faster results which allows to mitigate contamination risk at the moment it is detected. This projects lays the ground for further applications of RMM in the pharmaceutical manufacturing process.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Due to modern high-throughput technologies, large numbers of compounds are produced by parallel synthesis and combinatorial chemistry. The pharmaceutical industry therefore requires rapid and accurate methods to screen new drugs leads for membrane permeability potential in the early stages of drug discovery. Around 50 % of all investigational new drugs fail in pre-clinical and clinical phases of development due to inadequate absorption/permeation, distribution, metabolism, excretion and/or unacceptable toxicity. This may be decreased by applying in vitro screening methods early in the discovery process. Reliable in vitro models can be applied to determine permeation of the test compounds, which will help avoid the wasting of valuable resources for the development of drugs that are destined to fail in preclinical and clinical phases due to insufficient permeability properties. It is important to decide as early as possible on the most promising compound and physical formulation for the intended route of administration. With awareness of the increasing importance of in vitro models in the investigations of the permeability properties of drug compounds, this research project was specifically devoted to determine the suitability of our in vitro model to evaluate and predict drug permeability. A continuous flow-through diffusion system was employed to evaluate the permeability of nine different compounds/drugs with different chemical properties, across three biological membranes. The biological membranes chosen for the present study were human vaginal mucosa, human skin tissue and human small intestine mucosa. The continuous flow-through diffusion system was furthermore utilised to investigate the effects of de-epithelialisation of mucosal surfaces, chemical enhancers, temperature, permeant concentration and formulation on the permeability of the test compounds/drugs. The in vitro permeability information and data from the flow-through diffusion model were compared to in vitro and in vivo literature studies and drug profile. An in vitro model that is able to reliably predict in vivo data will shorten the drug development period, economise resources and may potentially lead to improved product quality. In this thesis research results are reported on the permeability of the mentioned biological membranes to the various chemical markers, including anti-HIV (human immunodeficiency virus) drugs. The permeability studies will be discussed in three sections: vaginal mucosa, skin tissue, small intestine mucosa. The results of the vaginal permeability studies showed that the three peptides (MEA- 5, MDY-19 and PCI) readily penetrated the vaginal mucosa. MDY-19 had a higher flux rate than MEA-5, commensurate with its smaller molecular size (weight). The surfactant enhanced the flux rate of MDY-19 approximately 1.3 times and decreased the lag time of the peptide. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. The permeability of 1 mM MDY-19 and PCI at 37 °C were significantly (p<0.05) higher than at 20 °C. At 37 °C the AUCs of the overall mean flux values of MDY-19 and PCI increased with concentration according to well-established diffusion theory. The experiments on the permeability of different terbinafine hydrochloride formulations through human skin demonstrated that the terbinafine hydrochloride formulations used in this study, readily diffused into the skin tissue. However, no flux values for any of the terbinafine hydrochloride formulations through the skin into the acceptor fluid were found. The mean terbinafine concentrations in the skin after 24 h exposure to the three commercial, terbinafine hydrochloride formulations were 3.589, 1.590 and 4.219 μg/ml respectively. The mean terbinafine concentration in the skin exposed to the 10 mg/ml PBS/Methanol solution was higher than those from the three commercial formulations. The results of the temperature study demonstrated that an increase of 5 ºC caused a significant increase in flux values of tritiated water across skin. The flux values for tritiated water across skin at 37 ºC were on average double those at a temperature of 32 ºC. The permeability of excised human small intestine mucosa to different oral dosage drugs was investigated over a 24 h period. The four drugs selected were zidovudine, propranolol hydrochloride, didanosine and enalapril maleate. They were selected as representative model compounds of drug classes 1 (high solubility, high permeability) and 3 (high solubility, low permeability) according to the Biopharmaceutics Classification System. The flux rates of the four chosen test drugs were influenced by the length of the experiment. Between the time periods 2-4 h and 4-6 h, zidovudine’s mean flux values across small intestine tissue were respectively 1.8 and 2.0 times higher than didanosine and 2.3 and 2.2 times higher than enalapril. Propranolol’s mean flux values were respectively 1.2 and 1.4 times higher than didanosine and 1.6 higher than enalapril during both the 2-4 and 4-6 h time periods. Between both the time periods 2-4 and 4-6 h AZT’s mean flux values were 1.4 times higher than propranolol and didanosine’s mean flux values were respectively 1.3 and 1.1 times higher than enalapril during the mentioned time periods. Class 1 drugs showed a significantly higher flux rate across the jejunal mucosa compared to the class 3 drugs and these results are in line with their Biopharmaceutics Classification System classification. The in vitro model has proved to be reliable to predict permeability of class 1 and 3 drugs and also showed correlation with human in vivo data. It seems that the in vitro flow-through diffusion model used in the present study have the potential to overcome some of the problems and limitations demonstrated by other in vitro techniques and may potentially serve as a future tool for pharmaceutical companies to predict the diffusion characteristics of new drugs and different formulations, across different biological membranes. Furthermore, it may serve as a prospective method for assessing the bioequivalence of alternative (generic) vehicles or formulations containing the same drug/compound.
AFRIKAANSE OPSOMMING: As gevolg van moderne hoë spoed tegnologie kan groot hoeveelhede middels vervaardig word deur ooreenkomende sintese en kombinasieleer chemie. Die farmaseutiese industrie benodig dus vinnige en akkurate metodes om nuwe geneesmiddels te evalueer t.o.v. membraan deurlaatbaarheid. Hierdie evaluasie moet verkieslik so vroeg moontlik in die geneesmiddel se ontwikkelingsproses geskied. Ongeveer 50 % van alle potensiële geneesmiddels misluk in pre-kliniese en kliniese fases van geneesmiddelontwikkeling. Die mislukte pogings kan toegskryf word aan onvoldoende absorbsie/deurlaatbaarheid, distribusie, metabolisme, ekskresie en/of onaanvaarbare middel toksisiteit. Dit is daarom belangrik om so vroeg moontlik in die geneesmiddelontwikkelingsproses te besluit op die mees belowende middel, asook die geskikte formulasie vir die spesifieke roete van toediening van die middel. Die farmaseutiese industrie benodig tans in vitro modelle met die potensiaal om die deurlaatbaarheid van geneesmiddels te bepaal en te voorspel. Betroubare in vitro modelle kan aangewend word om die deurlaatbaarheid van potensiële geneesmiddels te toets. Sodoende sal die onnodige uitgawes op die ontwikkkeling van geneesmiddels wat in elk geval later gaan faal in pre-kliniese en kliniese fases van geneesmiddelproewe a.g.v. deurlaatbaarheidseienskappe, vermy word. Hierdie navorsingsprojek was dus spesifiek onderneem om die waarde en toepaslikheid van ‘n in vitro deurlopende-vloei perfusie model te ondersoek. Die model se potensiaal om geneesmiddels se deurlaatbaarheid en absorpsie te voorspel was geëvalueer. Die deurlopende-vloei perfusie apparaat was gebruik om die deurlaatbaarheidsvloede van drie verskillende biologiese membrane t.o.v. nege chemiese stowwe (MEA-5, MDY-19, PCI, terbinafien hidrochloried, getritieerde water, zidovudien, propranolol, hidrochloried, didanosien, enalapril maleaat) te bepaal. Die drie biologiese membrane wat gebruik was, was vaginale weefsel, vel en klein intestinale weefsel. Al drie weefsel tipes was van menslike oorsprong. Die deurlopende-vloei perfusie apparaat was ook gebruik om die effek wat verwydering van die mukosa se epiteellaag op deurlaatbaarheidsvloede het, te ondersoek. Verder was navorsing gedoen op die effek van temperatuur en die konsentrasie en formulasie van die toetsmiddels op hulle diffusie vloedwaardes. Daar was ook gekyk na die invloed van ander chemiese stowwe op die toetsmiddels se diffusie vloedwaardes. Die in vitro deurlaatbaarheidsinformasie en -gegewens was vergelyk met ander in vitro en in vivo literatuurstudies en geneesmiddel databasisse. ‘n In vitro model wat in staat is om in vivo resultate betroubaar te voorspel, het die potensiaal om die tyd wat dit neem om geneesmiddels te ontwikkel, te verkort, finansiële uitgawes te besnoei en om geneesmiddelkwaliteit te verseker. In die tesis word dan die resultate gerapporteer van die deurlaatbaarheidsvloede van die verskillende tipes weefsel ten op sigte van verskeie chemiese stowwe, insluitende anti-MIV (menslike immuniteitsgebreksvirus) middels. Die deurlaatbaarheidstudies word bespreek in drie afdelings: vaginale mukosa, vel en klein intestinale mukosa. Die resultate van die deurlaatbaarheidstudies op die vaginale weefsel dui daarop dat die drie peptiede (MEA-5, MDY-19 and PCI) die vaginale mukosa goed penetreer. Soos verwag, het MDY-19 hoër diffusie vloedwaardes as MEA-5 gehad. Dit kan toegeskryf word aan MDY-19 se kleiner molekulere grootte (gewig). Surfaktant het die diffusie vloedwaardes van MDY-19 1.3 keer vergroot en het ook die tyd na vaste vlak verminder. Die verwydering van die vaginale epiteel het die diffusie vloedwaardes van die peptiede verhoog en mag dus dui op die vinniger opname van peptiede en moontlike ander mikrobisiede in vivo, wanneer die belyning van die epiteel onderbreek. Die deurlaatbaarheid van 1 mM MDY-19 en PCI by 37 °C was satisties beduidend (p<0.05) hoer as teem 20 °C. Die area onder die kurwe (AOK) van die gemiddelde vloedwaardes van MDY-19 en PCI by 37 °C, het toegeneem met ‘n toename in die konsentrasie van hierdie peptiede. Die toename vloedwaardes ondersteun dus die alombekende diffusie teorie. Die transdermale diffusie eksperimente van verskillende terbinafien formulasies het getoon dat terbinafien geredelik vrygestel word vanuit hierdie formulasies na die vel. Geen terbinafien vloedwaardes, van enige van die formulasies, was egter gevind in die ontvangselle van die deurlopende-vloei perfusie apparaat nie. Die gemiddelde terbinafien konsentrasies in die vel na 24 h se blootstelling aan drie kommersiële terbinafien hidrochloried formulasies was onderskeidelik 3.589, 1.590 en 4.219 μg/ml. Die gemiddelde terbinafien konsentrasie in die vel wat aan 10 mg/ml PBS/metanol blootgestel was, was hoër as die konsentrasies in die vel wat aan die drie kommersiële formulasies blootgestel was. Die resultate van die temperatuurstudie op vel het aangetoon dat ‘n temperatuur toename van 5 ºC ‘n statisties beduidende toename in vloedwaardes van getritieerde water oor vel veroorsaak. Die vloedwaardes van die getritieerde water oor vel teen ‘n temperatuur van 37 ºC was gemiddeld dubbeld so veel as teen 32 ºC. Die deurlaatbaarheidsvloede van klein intestinale mukosa ten opsigte van verskillende geneesmiddels (wat oraal toegedien word) was ondersoek gedurende ‘n 24 h eksperiment. Die vier geneesmiddels wat gebruik was, was zidovudine, propranolol hidrochloried, didanosien en enalapril maleaat. Hierdie geneesmiddels is verteenwoordigers van die Biofarmaseutiese Klassifikasie Sisteem se klas 1 (hoë oplosbaarheid, hoë deurlaatbaarheid) en klas 3 (hoë oplosbaarheid, lae deurlaatbaarheid) geneesmiddels. Die vloedwaardes van die vier geneesmiddels het gewissel na aanleiding van die tydsverloop in die eksperiment. Zidovudien se gemiddelde vloedwaardes tussen 2-4 en 4-6 h was onderskeidelik 1.8 en 2.0 keer hoër as didanosien se gemiddelde vloedwaardes vir hierdie tyd periodes en onderskeidelik 2.3 en 2.2 keer hoër as enalapril se gemiddelde vloedwaardes. Tydens hierdie selfde periodes was propranolol se gemiddelde vloedwaardes 1.2 en 1.4 keer hoër as didanosien en vir beide periods 1.6 keer hoër as enalapril se gemiddelde vloedwaardes. Gedurende beide genoemde tyd periodes was zidovudien se gemiddelde vloedwaardes 1.4 keer hoer as propranolol en didanosien se gemiddelde vloedwaardes was onderskeidelik 1.3 en 1.1 keer hoër as enalapril tydens 2-4 en 4-6 h. Die klas 1 geneesmiddels het statisties beduidende hoër vloedwaardes gehad as die klas 3 geneesmiddels. Hierdie resultate stem ooreen met die geneesmiddels se Biofarmaseutiese Klassifikasie Sisteem klassifikasie. Dit wil dus voorkom asof die in vitro model wat gebruik was in die studie, gebruik kan word om die deurlaatbaarheidsvloede van klas 1 en 3 te voorspel. Die resultate van hierdie studie stem ooreen met ander in vivo studies. Dit wil voorkom asof die in vitro deurlopende-vloei perfusie apparaat die potensiaal het om sommige van die probleme en tekortkominge van ander in vitro modelle te oorkom en dat dit moontlik die potensiaal het om die diffusie-eienskappe van nuwe geneesmiddels en verskillende formulasies oor verskillende biologiese membrane te voorspel. Die model kan verder moontlik dien as ‘n potensiële toestel om biogelykbaarheid van alternatiewe (generiese) formulasies, wat dieselfde geneesmiddel/chemiese stof bevat, te bepaal.

Title: Treatment of CMV Vitritis in a Preterm Newborn Author’s Section: Remil Simon1, Darshan Shah1, Peter Blosser1, Demetrio Macariola1, Jeffrey Carlsen2 1.Department of Pediatrics, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 2.Johnson City Eye Clinic, Johnson City, TN Body: Cytomegalovirus (CMV) infection in the neonate is an infrequent occurrence in the developing world, and observing the symptoms of ocular CMV infection such as vitritis is rare. Treating CMV infection promptly is necessary to prevent mortality and potential neurological deficits including blindness and hearing loss. We encountered a preterm infant presenting with CMV sepsis immediately after birth. Our question was: will the current standard of treatment for CMV sepsis prevent CMV ocular infection? With our method of treatment, we followed the current standard of treatment for CMV infection by administering intravenous Gancyclovir for 6 weeks and oral Valgancyclovir for 6 months. Despite using the standard treatment to prevent neurological sequelae, the patient developed CMV vitritis and retinitis bilaterally. Although the treatment did not prevent CMV ocular infection, the severity of CMV retinitis and vitritis improved with treatment, and full resolution of vitritis was noted by day of life 61.

Nanotechnology has become the most advanced type of drug delivery system within the last decade. This advancement shifted the focus on small carriers to increase the efficiency of the drugs. Among these, gold nanoparticles (GNPs) were found to have profound biomedical applications. In current research, kanamycin embedded GNPs were prepared in a single step, single phase, and bio-friendly (green synthesis) procedure. The synthesized Kanamycin-GNPs (Kan-GNPs) were spherical in shape and had a size range of 15 ± 3 nm. The chosen kanamycin is an aminoglycosidic antibiotic that is isolated from Streptomyces kanamyceticus. These special antibiotic GNPs are further characterized using several analytical methods like Transmission Electron Microscopy (TEM), Energy Dispersive Spectroscopy (EDS), Fourier Transform Infrared Spectroscopy (FTIR), and Ultra-Voilet/Visible spectroscopy (UV/Vis spectroscopy). The following research is a direct bio-friendly embedment of an antibiotic agent on the surface of the GNPs without any secondary capping agent or surface modification procedures.

Natural product drug discovery has traditionally been the corner stone of medicine having provided cures to many of today's most common diseases. In particular, antibiotics have revolutionised healthcare and extended human lifespan. However, since their introduction into the clinic, resistance to these drugs has arisen. With the number of new antibiotics being discovered in recent years declining, and fewer drugs making it past clinical trials, we have reached the point where antibiotic resistant infections have become common place and a serious threat to health and society. There is now an urgent requirement for the discovery of new antibiotics and in particular those with unexploited mode of action. This thesis details the different areas of natural product drug development from discovery through to analogue generation. In Chapter one, the history of natural products as therapeutics is explored with a particular focus on antibiotics and how resistance arises against these agents. It outlines why the discovery of new antibiotics is so important and new methods used to facilitate this search. Chapter two follows with the development of a screening platform for antibiotic induction, using the model Streptomyces; Streptomyces coleiolor M145. A variety of culture additives are explored for their ability to induce secondary metabolism production. Chapter three then details the sampling and identification of microbes from a pseudo-marine environment and their screening for their ability to produce secondary metabolites with antibiotic properties. The second half of this thesis centres on the non-ribosomal peptide echinomycin. Collaborators Aquapharm supplied the marine derived strain AQP-4895, capable of producing echinomycin. Chapter four details the establishment of AQP-4895 culturing conditions and the shift observed in production profile. Next Chapter five looks at producing echinomycin analogues through precursor directed biosynthesis. A range of halogenated quinoxaline carboxylic acids are synthesised and fed to AQP-4895, and the respective echinomycin analogues monitored by LC-MS. Chapter Six then aims to direct biosynthesis of the halogenated analogues, using mutasynthesis. Due to the lack of genetic data available surrounding the strain, an unusual approach was taken, using iPCR to create a template for homologous recombination.

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

El problema de la resistència bacteriana ha esdevingut un problema de salut pública global. Cada cop apareixen més soques bacterianes resistents a dos o més fàrmacs i és especialment preocupant la manca d'antibiòtics en el mercat per a combatre les infeccions que aquests provoquen. És per això que un dels grans objectius del descobriment de nous fàrmacs antibiòtics és la recerca de nous compostos capaços de combatre aquests microorganismes. Els pèptids antimicrobians són una classe d'antibiòtics que en el darrers anys han despertat interès degut al seu particular mecanisme d'acció: actuen principalment sobre la membrana bacteriana, i són menys susceptibles de generar resistència per part del bacteris. Destaquem entre aquests pèptids antimicrobians, la polimixina, antibiòtic natural produït pel Bacillus polymyxa. És un compost antibacterià actiu contra bacteris Gram-negatius, però que presenta l'inconvenient de ser tòxic a nivell renal i neuronal. En aquesta tesi es descriu el disseny, la síntesi i l'activitat d'anàlegs de polimixina. Es va simplificar l'esquelet peptídic del producte natural, de manera que fos mes assequible la síntesi química dels anàlegs, la preparació a gran escala, i una potencial reducció de toxicitat. Les modificacions que es van dur a terme van ser substituïr l'enllaç lactàmic que tanca el cicle, per un enllaç disulfur, l'àcid gras natural ramificat, es va substituir per un de lineal, i es van fer substitucions d'alguns aminoàcids per observar l'efecte que provocaven en l'activitat bacteriana. També es van sintetitzar anàlegs retroenantiòmers, per tal de veure la implicació de l'esquelet peptídic en la interacció amb les membranes, i finalment es va preparar un compost conjugat d'un anàleg de polimixina i l'espermina, una poliamina de cadena llarga. Es va estudiar la seva activitat antibiòtica in vitro amb assajos de concentració mínima inhibitòria (CMI). I posteriorment es van realitzar estudis d'activitat biofísica, realitzant assajos amb models de membrana bacteriana, com són els liposomes i les monocapes lipídiques. Aquesta tesi demostra que és possible simplificar l'estructura de la polimixina, obtenint-se compostos antibiòtics actius in vitro que mostren propietats biofísiques similars a les del producte natural.
The problem of bacterial resistance has become a global public health issue. Bacterial strains are becoming resistant to existing drugs and it is particularly worrying lack of antibiotics on the market to fight the infections they cause. That is why one of the major goals of antibiotic drug discovery today is the search for new compounds capable of combating these microorganisms. Antimicrobial peptides are a class of antibiotics that have raised particular interest these recent years due to its mechanism of action: they act primarily on the bacterial membrane, and are less likely to generate resistance by the bacteria. Among these antimicrobial peptides, the polymyxins, natural antibiotic produced by Bacillus polymyxa takes special interest for us. This antibacterial compound is active against Gram-negative bacteria, but it is nephrotoxic an neurotoxic. This thesis describes the design, synthesis and activity of polymyxin analogues. We report a simplified peptide structure, with a chemically accessible scaffold and upscalable, that might show potential toxicity reduction. The modifications that were carried out were substitution of the lactam bond that closes the heptapeptide cycle by a disulphide bond; branched natural fatty acid, was replaced by a linear one, and they were made some amino acid substitutions in order to observe the effect in the bacterial activity. We also synthesize analogues retroenantiomers, to see the involvement of the peptide scaffold when binding to bacterial membranes, and finally it was prepared a conjugate compound of an analogue of polymyxin and spermine, a long chain polyamine. We carried out assays of minimum inhibitory concentration (MIC) in vitro and also studies of biophysics activity, conducting tests with bacterial membrane models, such as liposomes and lipid monolayers. This thesis shows that it is possible to simplify the structure of polymyxin, obtaining active compounds in vitro with biophysical properties that are similar to the natural product.

En 1940, le biochimiste anglais Donald Woods propose une explication du mode d'action des nouveaux sulfamides antibactériens : l'inhibition compétitive. Son collègue Paul Fildes fonde sur cette base une nouvelle approche de la chimiothérapie, revendiquée comme rationnelle, un programme pour la recherche de nouveaux médicaments. Cette thèse explore l'impact de la théorie des antimétabolites, comme elle sera appelée, dans la recherche biochimique et pharmaceutique. La première partie retrace son élaboration dans le contexte de l'école de biochimie anglaise, puis sa reprise aux États-Unis à la suite de travaux menés en parallèle sur les vitamines. La seconde partie est consacrée au développement de deux programmes de recherche distincts dédiés aux antimétabolites, illustrant les modalités et fortunes divergentes d'appropriation de cette approche rationnelle. Le premier est une collaboration modeste entre le biochimiste Henry McIlwain et la firme pharmaceutique Glaxo pendant la guerre au Royaume-Uni. Le second consiste en la mise en place du programme de George Hitchings et Gertrude Elion chez Burroughs Wellcome aux États-Unis, souvent considéré comme l'origine du rational drug design actuel. La théorie des antimétabolites correspond aussi bien à l'ambition d'obtenir des chimiothérapies spécifiques qu'à un ensemble de pratiques dans le quotidien du laboratoire
In 1940, the British biochemist Donald Woods put forward an explanation of the mode of action of the new antibacterial sulfa drugs, competitive inhibition. His colleague, Paul Fildes, developed this work into a new approach to chemotherapy, which he qualified as a rational programme for drug discovery. This dissertation explores the impact of the theory of antimetabolites, as it came to be known, in biochemical and pharmaceutical research. The first part traces its development in the context of the British school of biochemistry and its further expansion in the United States following parallel research on vitamins. The second part deals with the construction of two distinct research programmes dedicated to antimetabolites, each one illustrating a different way of following this rational approach and their varying consequences. The first one is a modest collaboration between the biochemist Henry McIlwain and the Glaxo pharmaceutical company during the war in the United Kingdom. The second one corresponds to the establishment of George Hitchings' and Gertrude Elion's programme at Burroughs Wellcome in the United States, often considered as the origin of today's rational drug design. The theory of antimetabolites simultaneously embodied both the ambition of attaining specific chemotherapies, and a set of practices in day-to-day laboratory work

Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences. This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities. The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies. The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

Discovery of new antifungal chemo-therapeutics for humans is limited by the large degree of conservation among eukaryotic organisms. In recent years, the histone acetyl-transferase Rtt109 was identified as the sole enzyme responsible for an abundant and important histone modification, histone H3 lysine 56 (H3K56) acetylation. In the absence of Rtt109, the lack of acetylated H3K56 renders yeast cells extremely sensitive to genotoxic agents. Consequently, the ability to sustain genotoxic stress from the host immune system is crucial for pathogens to perpetuate an infection. Because Rtt109 is conserved only within the fungal kingdom, I reasoned that Rtt109 could be a novel drug target. My dissertation first establishes that genome stability provided by Rtt109 and H3K56 acetylation is required for Candida albicans pathogenesis. I demonstrate that mice infected with rtt109 -/- cells experience a significant reduction in organ pathology and mortality rate. I hypothesized that the avirulent phenotype of rtt109 -/- cells is due to their intrinsic hypersensitivity to the genotoxic effects of reactive oxygen species (ROS), which are utilized by phagocytic cells of the immune system to kill pathogens. Indeed, C. albicans rtt109 -/- cells are more efficiently killed by macrophages in vitro than are wild-type cells. However, inhibition of ROS generation in macrophages renders rtt109 -/- and wild-type yeast cells equally resilient to killing. These findings support the concept that ability to resist genotoxic stress conferred by Rtt109 and H3K56 acetylation is a virulence factor for fungal pathogens and establish Rtt109 as an opportune drug- target for novel antifungal therapeutics. Second, I report the discovery of a specific chemical inhibitor of Rtt109 catalysis as the initial step in the development of a novel antifungal agent. We established a collaboration with the Broad Institute (Cambridge, MA) to perform a high-throughput screen of 300,000 compounds. From these, I identified a single chemical, termed KB7, which specifically inhibits Rtt109 catalysis, with no effect on other HAT enzymes tested. KB7 has an IC50 value of approximately 60 nM and displays noncompetitive inhibition regarding both acetyl-coenzyme A and histone substrates. With the genotoxic agent camptothecin, KB7 causes a synergistic decrease in C. albicans growth rate. However, this effect is only observed in an efflux-pump mutant, suggesting that this compound would be more effective if it were better retained intracellularly. Further studies through structure-activity relationship (SAR) modifications will be conducted on KB7 to improve its effective cellular concentration.

Growing resistance of bacteria towards antibiotics resulted in extensive research effort for development and application of new materials and techniques. Due to their unique properties, graphene quantum dots (GQDs) have attracted much attention and are a promising material with potential applications in many fields. One use of GQDs is as a photodynamic therapy agent that generates singlet oxygen. In this work, GQDs synthesized by focusing nanosecond laser pulses into a mixture of benzene and nickel(II) oxide were combined with methylene blue (MB) to eradicate Gram-negative Escherichia coli and Gram-positive Micrococcus luteus. Theoretical calculation of pressure evolution was calculated using the standard finite difference method. Detailed characterizations were performed with transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR), UV-Visible (UV-Vis), and photoluminescence (PL) spectra. Furthermore, singlet oxygen generation from MB-GQD mixture was investigated by measuring the rate of 9,10-anthracenediyl-bis(methylene) dimalonic acid photobleaching at 400 nm. Combining MB with GQDs caused enhanced singlet oxygen generation, leading to improved bacterial deactivation rate. The (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to determine if GQDs in dark conditions caused human cellular side-effects and affected cancer and noncancer cellular viability. We found that even high concentrations of GQDs do not alter viability under dark conditions. These results suggest that the MB-GQD combination is a promising photodynamic therapy agent that may be useful when antibiotics resistance is present.

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Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil.
O Instituto de Tecnologia em Fármacos (Farmanguinhos), unidade técnico-científica da Fundação Oswaldo Cruz, representa o maior laboratório farmacêutico oficial vinculado ao Ministério da Saúde brasileiro responsável pela produção de mais de um bilhão de medicamentos por ano. As legislações sanitárias vigentes definem que devem ser utilizadas instalações segregadas e dedicadas para a produção de medicamentos, como penicilinas, minimizando os riscos de contaminação cruzada, que consequentemente possam gerar danos à saúde. Por isso, Farmanguinhos possui, atualmente, uma área dedicada à produção de amoxicilina, assim como o sistema gerador de água purificada, que atende à referida fabricação, também é dedicado à mesma. A água é a matéria-prima de mais elevado volume empregada na produção farmacêutica, exercendo profundo impacto na qualidade do produto e na segurança do paciente. O controle microbiológico da qualidade da água utilizada em processos farmacêuticos torna-se fundamental, já que micro-organismos podem sobreviver e proliferar em sistemas de água, tornando-se fontes de contaminação microbiana e pirogênica. A identificação destes micro-organismos e as informações obtidas sobre os mesmos podem ser extremamente úteis na identificação da fonte de contaminação microbiana de um produto ou processo, alem de direcionar ações corretivas, se necessário. O objetivo deste trabalho foi avaliar a diversidade microbiana presente em sistema gerador de água purificada, dedicado à produção de penicilínicos, de Farmanguinhos. Para isto, as amostras de água coletadas a partir do ponto de entrada, ponto de água potável, e do ponto de saída do sistema, ponto de água purificada, foram analisadas microbiologicamente quanto à contagem de micro-organismos mesófilos e quanto à presença de patógenos: Escherichia coli; coliformes totais e Pseudomonas aeruginosa. As amostras foram analisadas entre janeiro e maio de 2013. Todas as colônias obtidas foram isoladas e submetidas à identificação, através de equipamento MALDI-TOF. Não foram observados resultados acima da especificação para ambos os pontos, no período analisado. Do ponto de água potável foram isoladas apenas três espécies bacterianas: Bacillus cereus, Delftia acidovorans e Acinetobacter sp. Uma diversidade microbiana maior foi isolada a partir do ponto de água purificada, como: Burkholderia sp, Delftia acidovorans, Stenotrophomonas maltophilia, Bacillus cereus, Pseudomonas oryzihabitans e Pseudomonas putida. A espécie Delftia acidovorans, associada a fenômenos de biocorrosão foi o micro-organismo mais encontrado, sendo identificado em cerca de 1/3 do total de isolados. O sistema de água de Farmanguinhos é sanitizado mensalmente com sanitizante à base de glutaraldeído. Pode-se concluir que a sanitização está sendo eficiente para manter a contagem total de micro-organismos mesófilos dentro dos limites especificados. No entanto, algumas ações podem ser tomadas para evitar a presença de biofilmes, o que aprimorará ainda mais a qualidade da água purificada gerada.
The Instituto de Tecnologia em Fármacos (Farmaguinhos), technical- scientific unit of Fundação Oswaldo Cruz, is the largest official pharmaceutical laboratory under the Brazilian Ministry of Health responsible for producing more than one billion drugs per year. The current sanitary laws define that segregated facilities must be dedicated and used for the production of drugs such as penicillin, thus the risk of serious damage to health, due to crosscontamination, can be minimized. Because of this, Farmanguinhos has a dedicated production of amoxicillin, as well as a purified water generator system that caters to such manufacturing is also dedicated to this area. Water is the raw material of the highest volume used in pharmaceutical production so that it has a profound impact on the product quality and patient safety. Therefore, the microbiological quality control of water used in pharmaceutical processes becomes crucial, since microorganisms can survive and proliferate in water systems, becoming sources of microbial and pyrogenic contamination. The identification of these microorganisms and the information obtained about them can be extremely useful in identifying the source of microbial contamination of a product or process, in addition to direct corrective actions, if necessary. The objective of this study was to evaluate the microbial diversity present in the purified water generator system, dedicated to the area of manufacturing penicillin-based drugs of Farmanguinhos. To aim this objective, water samples collected from the entry point of the system, point of drinking water, and the point of departure, point of purified water, were analyzed microbiologically for the count of mesophilic and for the presence of pathogens: Escherichia coli; coliforms and Pseudomonas aeruginosa, respectively. The samples were analyzed between January and May of 2013. All colonies obtained were isolated and subjected to identification by MALDI - TOF equipment. No results above specification for both points were observed in the analyzed period. From the point of drinking water only three bacterial species were isolated: Bacillus cereus, Acinetobacter sp and Delftia acidorovans. A higher microbial diversity was observed from the purified water source, such as Burkholderia sp, Delftia acidovorans, Stenotrophomonas maltophilia, Bacillus cereus, Pseudomonas putida and Pseudomonas oryzihabitans. The species Delftia acidovorans associated with biocorrosion phenomena was the microorganism most frequently found and was identified in about 1/3 of the total isolates. The water system of Farmaguinhos water is sanitized monthly with sanitizing agent comprising glutaraldehyde. It can be concluded that sanitization is being effective to maintain the total count of mesophilic within the specified limits. However, some actions can be taken to avoid the presence of biofilms, which will improve even more the quality of purified water generated.

High-throughput drug screening (HTS) in live cells is often a vital part of the preclinical anticancer drug discovery process. So far, two-dimensional (2D) monolayer cell cultures have been the most prevalent model in HTS endeavors. However, 2D cell cultures often fail to recapitulate the complex microenvironments of in vivo tumors. Monolayer cultures are highly proliferative and generally do not contain quiescent cells, thought to be one of the main reasons for the anticancer therapy failure in clinic. Thus, there is a need for in vitro cellular models that would increase predictive value of preclinical research results. The utilization of more complex three-dimensional (3D) cell cultures, such as multicellular tumor spheroids (MCTS), which contain both proliferating and quiescent cells, has therefore been proposed. However, difficult handling and high costs still pose significant hurdles for application of MCTS for HTS. In this work, we aimed to develop novel assays to apply MCTS for HTS and drug evaluation. We also set out to identify cellular processes that could be targeted to selectively eradicate quiescent cancer cells. In Paper I, we developed a novel MCTS-based HTS assay and found that nutrient-deprived and hypoxic cancer cells are selectively vulnerable to treatment with inhibitors of mitochondrial oxidative phosphorylation (OXPHOS). We also identified nitazoxanide, an FDA-approved anthelmintic agent, to act as an OXPHOS inhibitor and to potentiate the effects of standard chemotherapy in vivo. Subsequently, in Paper II we applied the high-throughput gene-expression profiling method for MCTS-based drug screening. This led to discovery that quiescent cells up-regulate the mevalonate pathway upon OXPHOS inhibition and that the combination of OXPHOS inhibitors and mevalonate pathway inhibitors (statins) results in synergistic toxicity in this cell population. In Paper III, we developed a novel spheroid-based drug combination-screening platform and identified a set of molecules that synergize with nitazoxanide to eradicate quiescent cancer cells. Finally, in Paper IV, we applied our MCTS-based methods to evaluate the effects of phosphodiesterase (PDE) inhibitors in PDE3A-expressing cell lines. In summary, this work illustrates how MCTS-based HTS yields potential to reveal and exploit previously unrecognized tumor-specific vulnerabilities. It also underscores the importance of cell culture conditions in preclinical drug discovery endeavors.

La planification automatique des traitements de radiothérapie n´ecessite un calcul très rapide de la dose délivrée au patient. Nous proposons de réaliser ce calcul en segmentant le fantôme du patient en mailles homogènes, et en associant aux mailles des projections vers des distributions précalculées en milieux homogènes, ainsi que des pondérations gérant les hétérogénéités. Le calcul de dose se fait en deux étapes. La première étape concerne les mailles : les projections et pondérations y sont paramétrées en fonction de critères physiques et géométriques. La seconde étape concerne les voxels : la dose y est calculée en évaluant les fonctions préalablement associées à leur maille. Cette méthode est très rapide, notamment quand le nombre de points d'intérêt est limité (quelques centaines), les résultats étant dans ce cas obtenus en moins d'une seconde. Avec de telles performances, la planification automatique des traitements de radiothérapie devient parfaitement envisageable.

The use of plant materials for the treatment of various diseases is very common in African countries. As traditional medicine used by the rural people does not always have a proper scientific basis, research programmes have to be undertaken to evaluate their therapeutic efficacy and safety. In traditional African medicine various Bulbine species are used to treat a number of conditions including sexually transmitted diseases, wound infections, dysentery and urinary tract infections. The Bulbine species belong to the family Asphodelaceae. There are over fifty South African Bulbine species and they are mostly herbs. Their leaves are evergreen and succulent in appearance. Bulbine species have thick fleshy tuberous roots, are easy to grow, are able to withstand drought and heat and are able to grow in poor soil. There is very little documented information on the antimicrobial activity and chemical properties of the Bulbine species. Therefore research programmes of this nature have to be undertaken. Various Bulbine species, viz., B. natalensis Bak, B. frutescens Willd (yellow flowers), B. narcissifalia Salm Dyck, B. abyssinica A Rich and B. frutescens Willd (orange flowers) were collected. The plants were washed with tap water, air dried and separated into the different components. Each component was cut into small pieces and immersed in methanol: dichloromethane (1:1, v/v) for extraction. The organic solvent was decanted from the plant material and evaporated under reduced pressure. The resultant crude extracts were stored in glass vials in the freezer. In addition, the roots, stems and leaves of B. natalensis and B. frutescens (yellow flowers) were extracted aqueously. The crude organic and aqueous were subjected to various tests to evaluate their antimicrobial and cytotoxic potential. To evaluate their antibacterial activities, the Disk Diffusion and Bore Well Methods were employed. The crude extracts were tested against various pathogens implicated in wound and urinary tract infections and dysentery. In these experiments the Disk Diffusion Method produced better results than the Bore Well Method. The crude organic and aqueous extracts were found to be effective against many of the bacteria used in this study including K. pneumoniae, S. aureus, S. typhi and S. flexneri which are considered to be troublesome pathogens. The TLC bioassay was employed to evaluate the antifungal potential of the various crude extracts against Aspergillus and Penicillium and the Disk Diffusion and Bore Well methods were used to evaluate the antifungal potential of C. albicans. The Bulbine species displayed no antifungal activity against Penicillium and limited antifungal activity against Aspergillus. The two method used to evaluate the antifungal activity of. C albicans was chosen because C. albicans grows in a similar manner to bacteria on solid and liquid culture media. Only the root extracts of the two B. frutescens varieties were inhibitory to C. albicans. The Brine Shrimp Bioassay was used to ascertain the cytotoxic potential of the crude extracts. The majority of the extracts were cytotoxic at the most concentrated dilution (i.e., dilution 1) but not cytotoxic at the lower dilutions. The only extracts that were not cytotoxic at the most concentrated dilution were the organic extract of the root of B. frutescens (yellow flowers), the organic extract of the root of B. narcissifolia and the organic extract of the leaf of B. abyssinica. TLC and column chromatography was carried out to evaluate the chemical composition of the Bulbine species. The TLC indicate that this technique could be a valuable tool in identifying the different species in the genus Bulbine. Column chromatogram was carried out on the extract which displayed a significant amount of antibacterial activity against the bacteria used in this study. The stem extract of B. natalensis was chosen for further analysis. The stem extract was fractitioned into different fractions but unfortunately none of the chemical component could be identified. According to the results obtained in this study, there is considerable scope for further studies of this genus.
Thesis (M.Med.Sc.)-University of Durban-Westville, 2000.

Important variables such as the cell mass and the product concentrations cannot be measured on-line in bioprocesses because of the limitations in sensor technology. In this dissertation crucial relationships which tie the measured variables to the unmeasured ones thereby overcoming these problems, are developed from a Systems Theory perspective. A successful application is demonstrated with an actinomycete fermentation to produce Spectinomycin A mathematical technique called the External Differential Representation demonstrates how a process can be reconstructed from only a part of the data. The energy demand in secondary metabolite fermentations vary between the growth and the production phases. This variation in energy requirements, which depends on the particular metabolite and the biosynthetic pathway for its production, is reflected in the air flow rate data at constant dissolved oxygen. From the information contained in the airflow rate and the substrate concentration data, the External Differential Representation enables a continuous prediction of Spectinomycin concentration Stringent requirements of bioprocesses demand the application of superior controllers with the capability of regulating the process within a narrow range about the set point. To achieve this, a Model-based Geometric Algorithm is developed from the concepts of the relative order of a system and geometric transformations. It implemented for controlling the dissolved oxygen and the pH in Spectinomycin fermentation. This algorithm uses the geometric information to adapt to the process changes and computes the control action by minimizing the anticipated error that would occur one sampling time ahead The experimental data show that more than one bioactive metabolite comprises the final product. An Activation-Reaction-Inhibition formulation proposed here describes the production of a single metabolite found in the final product. The advantages of the Activation-Reaction-Inhibition structure include higher parameter sensitivity and easy extension for describing multiple metabolite effects. The exponential term in the Activation-Reaction-Inhibition kinetics is responsible for these attributes which are not present in the Monod or its related structures. Hence, the trends in the experimental data are well represented with a model that incorporates this Activation-Reaction-Inhibition feature thereby validating the hypothesis This application of the External Differential Representation, the Model-based Geometric Algorithm and the Activation-Reaction-Inhibition model in reconstructing, monitoring and controlling, and describing bio-kinetics is successfully demonstrated in Spectinomycin fermentation
acase@tulane.edu

A major factor in the ability of Pseudomonas fluorescens Pf-5 to act as a biological control agent is its production of antibiotics, including pyoluteorin (PLT), 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin (PRN). The data provided in this thesis demonstrate that the presence of any of these antibiotics in the extracellular milieu affects production of that same antibiotic, as well as others, by Pf-5. Amending the growth medium with antibiotics had multiple effects on secondary metabolism in Pf-5. i) PLT positively regulated its own production, ii) 2,4-DAPG positively regulated its own production. iii) PLT suppressed 2,4-DAPG production. iv) 2,4- DAPG inhibited PLT production. v) PLT suppressed transcription of a heterologous ferric-pyoverdine uptake gene. vi) PRN exerted a slight inhibitory effect on PLT gene transcription and production. PLT autoinduction by Pf-5 was extensively characterized, and was shown to require concentrations of exogenous PLT in the nanomolar range. These low concentrations are comparable to those of many molecules proposed to function in signaling roles. PLT served as a signal between distinct populations of cells within the rhizosphere, where it prompted autoinduction by those cells. Aside from effects of Pf- 5 antibiotics on one another, I also described the positive effect of exogenous PLT on expression of a set of transport genes flanking the PLT biosynthetic gene cluster. Sequence data and experimental evidence suggests that these genes encode a transport apparatus for PLT. The deduced amino acid sequences for four adjacent open reading frames together resemble Type I secretion apparatuses, which typically function in transport of proteins rather than secondary metabolites. The intact transporter genes are necessary for optimal PLT production. Taken together, the data from the studies described herein demonstrate that i) the production of PLT by Pf-5 can affect the production of PLT by neighboring cells, and ii) PLT and other exogenous secondary metabolites have both autoregulatory and cross-regulatory effects in culture. Because Pf-5 derivatives engaged in PLT crossfeeding in the rhizosphere, it is likely that cross-feeding occurs for other secondary metabolites as well. Thus, production of an antibiotic by one cell can profoundly affect secondary metabolism in neighboring cells occupying natural habitats.
Graduation date: 2004

Large amounts of human pharmaceutical products are consumed worldwide. Many drugs and their metabolites, referred to as pharmaceutically active compounds (PhACs), are not fully metabolized prior to household discharge resulting in their common occurrence in wastewater treatment plants (WWTPs). In most instances, WWTPs present the first treatment opportunity for removing PhACs and preventing significant environmental exposure. Because most municipal WWTPs rely on the microbial component of the activated sludge process, there is a need to estimate the influence of PhACs in wastewater influent on the activated sludge microbial communities and the treatment performance of WWTPs. The objective of this dissertation was to determine the impact of selected PhACs (i.e., ketoprofen, naproxen, clofibric acid, carbamazepine and gemfibrozil) on activated sludge microorganisms and key individual microbial species in domestic wastewater treatment. Analyses were performed in batch reactors initially and then in laboratory-scale sequencing batch reactors (SBR) which mimic WWTP operations. Ammonia oxidizing bacteria (AOB) were selected as indicator organisms because of their importance in wastewater treatment and demonstrated sensitiveness to toxic compounds.

The batch experiments results suggested that microbial growth inhibition was correlated to organic loadings. In the presence of 0.2% (v/v) ethanol, significant inhibition, ranging from 34 to 43%, was observed for all PhACs other than clofibric acid.

Nitrification inhibition studies using Nitrosomonas europaea, a model AOB strain showed that ketoprofen, naproxen, carbamazepine and gemfibrozil inhibited nitrite production. The corresponding maximum nitrification inhibition rates were 25, 29, 22 and 26%, respectively. Inhibition was shown to increase with PhAC concentration for concentrations greater than 0.1 µM. Results from membrane integrity tests suggest that the inhibition may be due to the disturbance of the cell membrane by PhACs and such inhibition was shown to be irreversible.

Even though PhACs were shown to inhibit the nitrification rate in pure culture studies, the performance of SBRs exposed to individual PhACs was not adversely affected neither in terms of COD nor ammonia removal. Microbial fingerprinting for both total bacteria and AOB confirmed that no significant shifts occurred when microbial communities were exposed to PhACs. However, some PhACs introduced in binary mixture were found to both inhibit the nitrification of N. europaea as well as the performance of SBRs. The mixture composed of 0.5 μM ketoprofen and 0.5 μM naproxen showed significant inhibition (25%) on the nitrite production of N. europaea although neither 0.5 μM ketoprofen nor 0.5 μM naproxen had significant effect when presented alone. Similarly, both COD and ammonia removal were significantly impacted by binary mixtures of PhACs. These results suggest that mixture effects can play an important role in an overall treatment's nitrification potential and this phenomenon should be further investigated.

Dissertation

This study examined temporal variation of pharmaceutical concentrations in two streams with differing land uses: 1) a suburban stream with combined sewer overflow point sources; and, 2) a rural stream influenced by septic systems and agricultural runoff. Sites were sampled monthly for pharmaceutical concentrations and stream physiochemical parameters. Pharmaceuticals were frequently detected in both the urban and agricultural stream with the highest concentrations measured during winter. Across sites, water column dissolved oxygen concentrations positively correlated with several pharmaceuticals suggesting microbial activity is important in pharmaceutical persistence. Potential for degradation of pharmaceuticals as a carbon or nitrogen source by stream sediment microbial communities was also estimated using pharmaceutical-amended basal salt media incubated under different temperature and ultraviolet (UV) light treatments. Under 4°C incubation, caffeine and acetaminophen were the most recalcitrant compounds whereas cotinine was the most labile. Under UV-B exposure, cotinine and sulfamethoxazole were the most recalcitrant compounds whereas ibuprofen was the most labile.
Temporal variation of pharmaceuticals in an urban and agriculturally influenced stream -- Degradation potential of six pharmaceuticals by sediment microbial communities.
Department of Biology